ABSTRACT Neuroblastoma (NB) is an aggressive childhood cancer in which the MYC homologue MYCN acts as an oncogene typically activated to drive tumor progression. NB is a lethal tumor and the survival rate of children with high-risk (stage IV) disease is dismal despite an intense treatment plan that includes a plethora of high- dose chemotherapies, surgery, stem cell transplant, radiation, and antibody therapy. MYCN and ornithine decarboxylase (ODC) are frequently deregulated in NB, and ODC-dependent polyamines control p27/Rb- regulated tumor progression. MYC(N) is a direct activator of ODC and ODC is a validated drug target in NB and other MYC(N)-driven tumors. Difluoromethylornithine (DFMO) is the only ODC inhibitor in the clinic today. DFMO is a FDA-approved anti-protozoan drug (trypanosomiasis) that is effective in the chemoprevention of colon cancer and that has entered clinical studies for children with relapsed NB. Unfortunately, DFMO is required at very high doses due to its short half-life, high solubility, and fast renal elimination/clearance. Therefore, the search for a new ODC inhibitor with higher potency and superior pharmacological profile is warranted. We have previously discovered that the natural product phaseolotoxin (PT) inhibits ODC and exhibits antiproliferative activity in cancer cells. The central hypothesis of this proposal is that novel PT- inspired PSorn-analogs designed for this study are more potent ODC inhibitors than PT or DFMO and, therefore, more effectively inhibit ODC-dependent NB tumor growth in vivo. The objective of this proposal is to identify the most active PSorn-analog with best PK characteristics and confirm its validity as a novel therapeutic agent against NB. In Aim 1 we will chemically synthesize a series of PSorn-analogs for biological & preclinical investigation. We will prepare 17-20 (linear & cyclic) PSorn-analogs. In Aim 2 we will test the hypothesis that these novel PSorn-analogs are potent ODC inhibitors in vitro. In Aim 3 we will test the efficacy of PSorn-analogs in cell-based oncology models and tumor xenograft studies. Active analogs will be selected through a testing funnel that includes: ODC activity and enzyme kinetic assays, NB cell-based oncology models using a panel of 24 well-characterized (MYCN amplified and MYCN non-amplified) NB cell lines with various ODC expression levels, in vitro ADME, and NB tumor xenograft efficacy studies in mice with DFMO- resistant NB cells. Isothermal calorimetry (ITC) and X-ray crystallization will be employed with most active PSorn-analogs to verify ODC binding domains and to aid further SAR-based analog optimization. In summary, these studies will provide the solid groundwork for further preclinical development of an entirely new class of ODC-targeted therapeutic agents with applications in the treatment and prevention of (DFMO-resistant) NB, colon cancer, and other MYC(N)-driven cancer types.